The major objective is to understand the mechanism by which estrogens as in pregnancy or oral contraceptive use, decrease the biliary excretion of drugs. These studies will focus on the transport across the hepatocyte of organic anions, a category to which belong many drugs, endogenous compounds, such as bilirubin and steroid hormones, and/or glucuronide and sulfate conjugates. Estradiol-17Beta(Beta-D-glucuronide) (E217G) will serve as a model organic anion. Two aproaches will be used to probe the organic anion transport system. (1) Ligand binding studies using 3H-E2-17G will identify specific binding sites, postulated to represent organic anion carriers, and provide information regarding the number of the binding sites (B-max) and the affinities (KD) of various organic anions for the sites. (2) Transport studies in vesicles will provide information regarding the driving forces for translocation of 3H-E217G. Membrane preparations used are hepatic basolateral plasma membranes which are relatively free of contamination with canalicular membranes and canalicular plasma membranes which are free of basolateral membranes. Binding studies will utilize a microcentrifugation technique to separate bound from free ligand, and thus minimize dissociation of bound ligand. Data will be analyzed using the computer program "ligand". Uptake and efflux will be quantitated in the presence of Na+- and K+-gradients using a filtration method. Inhibition studies will characterize the KD and Ki values in binding and transport studies respectively for a wide range of organic anions, (e.g., acetaminophen glucuronide and sulfate, bilirubin) in order to descibe the substrate specificities of these systems. The rank orders of potencies of inhibitors will be compared in binding studies and transport studies; the same rank order of potency will be used as evidence supporting the postulate that the binding site(s) represent the organic anion carrier(s). Likewise, rank orders of potency of inhibitors will be compared for basolateral and canalicular membrane preparations to determine if these sites/carriers are the same. Efflux studies in basolateral membranes will test the hypothesis that organic anions (e.g., drug glucuronides) are transported from liver to blood. The effects of estrogen treatment and pregnancy on binding (Kd, B-max) and kinetic (Km, V-max) parameters will be defined. Finally, the effect of the cholestatic E217G and the noncholestatic estradiol-3-glucuronide on transport of taurocholate in basolateral and canalicular vesicles will be examined.